The long term goals of this research are to elucidate the catalytic mechanisms of enzymes that use tetrahydrofolate derivatives as cofactors, and to study the regulation of one carbon metabolism. This research will emphasize mechanistic studies of two enzymes, cobalamin-dependent methionine synthase and serine hydroxymethyltransferase, that are potential targets for cancer chemotherapy, but for which no specific inhibitors are currently available. Strategies are proposed for the design of specific active site-directed inhibitors of each of these enzymes. Cobalamin- dependent methionine synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate (CH3-H4folate) to homocysteine, with the enzyme- bound cobalamin prosthetic group serving as an intermediary in the methyl transfer. We plan to determine the mode by which the substrate, CH3- H4folate, is activated for methyl transfer by using UV-visible and NMR spectroscopy to determine whether the substrate is protonated or oxidized in dead-end ternary complexes. We will investigate the mechanism by which the enzyme is inactivated by the commonly used anesthetic, nitrous oxide, and will attempt to design agents that are capable of inactivation of the enzyme by a similar mechanism. Related studies on the activation of methionine synthase by reductive methylation, and on the catalytic mechanism of a cobalamin-independent methionine synthase that catalyzes a closely similar reaction are also proposed. We will continue our studies on the catalytic mechanism of serine hydroxymethyltransferase, a pyridoxal phosphate-dependent enzyme that catalyzes the transfer of the beta-carbon of serine to H4folate to yield glycine and CH2-H4folate. These studies will attempt to elucidate the role of H4folate in the reaction mechanism and will explore the use of halogenated amino acids as potential inhibitors of catalysis. The availability of highly specific inhibitors for both methionine synthase and serine hydroxymethyltransferase will not only suggest approaches for chemotherapeutic targeting of these enzymes, but will also permit an assessment of the role of these enzymes in cellular metabolism.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM024908-23
Application #
6179257
Study Section
Special Emphasis Panel (NSS)
Program Officer
Ikeda, Richard A
Project Start
1978-04-01
Project End
2001-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
23
Fiscal Year
2000
Total Cost
$326,765
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Koutmos, Markos; Gherasim, Carmen; Smith, Janet L et al. (2011) Structural basis of multifunctionality in a vitamin B12-processing enzyme. J Biol Chem 286:29780-7
Matthews, Rowena G (2009) Cobalamin- and corrinoid-dependent enzymes. Met Ions Life Sci 6:53-114
Huang, Sha; Romanchuk, Gail; Pattridge, Katherine et al. (2007) Reactivation of methionine synthase from Thermotoga maritima (TM0268) requires the downstream gene product TM0269. Protein Sci 16:1588-95
Yamada, Kazuhiro; Strahler, John R; Andrews, Philip C et al. (2005) Regulation of human methylenetetrahydrofolate reductase by phosphorylation. Proc Natl Acad Sci U S A 102:10454-9
Evans, John C; Huddler, Donald P; Hilgers, Mark T et al. (2004) Structures of the N-terminal modules imply large domain motions during catalysis by methionine synthase. Proc Natl Acad Sci U S A 101:3729-36
Bandarian, Vahe; Matthews, Rowena G (2004) Measurement of energetics of conformational change in cobalamin-dependent methionine synthase. Methods Enzymol 380:152-69
Hondorp, Elise R; Matthews, Rowena G (2004) Oxidative stress inactivates cobalamin-independent methionine synthase (MetE) in Escherichia coli. PLoS Biol 2:e336
Bandarian, Vahe; Ludwig, Martha L; Matthews, Rowena G (2003) Factors modulating conformational equilibria in large modular proteins: a case study with cobalamin-dependent methionine synthase. Proc Natl Acad Sci U S A 100:8156-63
Kacprzak, Magdalena M; Lewandowska, Irmina; Matthews, Rowena G et al. (2003) Transcriptional regulation of methionine synthase by homocysteine and choline in Aspergillus nidulans. Biochem J 376:517-24
Dorweiler, Jeanne Sirovatka; Finke, Richard G; Matthews, Rowena G (2003) Cobalamin-dependent methionine synthase: probing the role of the axial base in catalysis of methyl transfer between methyltetrahydrofolate and exogenous cob(I)alamin or cob(I)inamide. Biochemistry 42:14653-62

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